Peptide bond formation is one of the central reactions of protein synthesis. It is catalyzed by the peptidyl transferase center located on the large ribosomal subunit. In spite of years of research, the composition, structure and function of peptidyl transferase remains obscure. One of the main obstacles for understanding organization and function of the peptidyl transferase center is the enormous complexity of the ribosome. Therefore, we propose a set of experiments whose goal is to isolate the elementary peptidyl transferase by significantly reducing complexity of the peptidyl transferase-active subribosomal particles. Subjection of 50S ribosomal subunits from thermophilic eubacterium Thermus aquaticus to extensive protein extraction procedures results in formation of particles (KSP particles) which, while containing 23S rRNA, 5S rRNA and only 8 ribosomal proteins, posses a high level of peptidyl transferase activity. The protein composition of KSP particles will be reduced by reconstituting rRNA with different combinations of proteins isolated from KSP particles. The minimal set of proteins required to support peptidyl transferase activity will be identified. Next, the importance of 5S rRNA for reconstituting active particles will be investigated. Preliminary studies indicated that 5S, which interacts with distant domains in 23S rRNA and whose presence is essential for in vitro assembly of functional peptidyl transferase, can be replaced by small drug molecules also interacting with the same two domains of 23S rRNA. The possibility of restoring high levels of peptidyl transferase activity of 5S-deficient particles reconstituted in the presence of antibiotics or small 5S rRNA fragments will be investigated. Next, extended stretches of 23S rRNA, non-essential for peptidyl transferase catalysis, will be deleted. These experiments will be based on a recent finding that functionally active large ribosomal subunits of T. aquaticus can be reconstituted with the in vitro transcribed 23S rRNA. The role of ribosomal proteins in peptidyl transferase activity maybe that of stabilizing the functional structure of catalytically-active rRNA. We will use a combination of rRNA mutagenesis, in vitro assembly and selection-amplification of RNA from subribosomal particles in order to select for rRNA mutations that, by stabilizing rRNA structure, may compensate for the lack of ribosomal proteins. An analogous approach will be used to find mutations compensating for the lack of 5S rRNA and/or the lack of extended segments of 23S rRNA in catalytically active particles. Once the minimalist peptidyl transferase is isolated, its architecture, detailed structure and functions will be analyzed. This information may not only clarify the mechanism of catalysis of peptide bond formation, but also provide important insights into organization and function of the protoribosome and evolution of translation apparatus. Understanding structure and function of the main catalytic center of the ribosome is indispensable for the rational design of new antibiotics and overcoming mechanisms of drug resistance.